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WO2004013357A2 - Sequences nucleotidiques specifiques a francisella tularensis et methodes de detection de francisella tularensis - Google Patents

Sequences nucleotidiques specifiques a francisella tularensis et methodes de detection de francisella tularensis Download PDF

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Publication number
WO2004013357A2
WO2004013357A2 PCT/US2003/024218 US0324218W WO2004013357A2 WO 2004013357 A2 WO2004013357 A2 WO 2004013357A2 US 0324218 W US0324218 W US 0324218W WO 2004013357 A2 WO2004013357 A2 WO 2004013357A2
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WO
WIPO (PCT)
Prior art keywords
pcr
francisella tularensis
nucleotide sequences
seq
sample
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PCT/US2003/024218
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English (en)
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WO2004013357A3 (fr
Inventor
Paula M. Mccready
Lyndsay Radnedge
Gary L. Andersen
Linda L. Ott
Thomas R. Slezak
Thomas A. Kuczmarski
Elizabeth A. Vitalis
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The Regents Of The University Of California
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Priority to AU2003269938A priority Critical patent/AU2003269938A1/en
Publication of WO2004013357A2 publication Critical patent/WO2004013357A2/fr
Publication of WO2004013357A3 publication Critical patent/WO2004013357A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • NUCLEOTIDE SEQUENCES SPECIFIC TO FRANCISELLA TULARENSIS
  • Francisella tularensis is the species of bacteria known to cause what is commonly known as Tuleremia, a serious and sometimes fatal disease. Since the attack on the World Trade Center in New York of September 11, 2001, there has been a growing concern that terrorists or rogue governments will use the Francisella tularensis bacterium as a weapon of mass destruction and instrument of terror. Since the events of September 11, 2001, the United States Government has been developing reliable methods and systems to detect the Francisella tularensis bacterium so that immediate and effective counter measures can be undertaken. The existing methods for detecting the Francisella tularensis bacterium are considered inadequate because of the higher than acceptable rate of false positive and false negative results.
  • An aspect of the invention includes the nucleotide sequences that are identified in SEQ ID NOs:4, 8, 12, 16, 20, 24, 28 and 32 that are specific to Francisella tularensis.
  • Another aspect of the invention includes a Forward Primer, the nucleotide sequences that are identified in SEQ ID NOs:l, 5, 9, 13, 17, 21, 25, and 29 and any primers that are derived from these nucleotide sequences.
  • a further aspect of the invention is a Reverse Primer, the nucleotide sequences that are identified in SEQ ID NOs:2, 6, 10, 14, 18, 22, 26 and 30 and any primers that are derived from these nucleotide sequences.
  • a further aspect of the invention is includes a Hybridization Probe, the nucleotide sequences that are .identified in SEQ ID NOs:3, 7, 11, 15, 19, 23, 27 and 31 and any probes that are derived from these nucleotide sequences.
  • This invention also includes a method for the detection of Francisella tularensis using the bacterium specific nucleotide sequence comprising: providing a sample in an environment that is suitable for isolating genomic DNA for amplification using PCR and under conditions suitable for hybridization with a least one group of nucleotides consisting or forward primer, a reverse primer and a hybridization probe and detecting the existence of Fracisella Tularensis specific nucleotide sequences by a nucleotide detection method, such as PCR and flurogenic 5' nuclease PCR assay, wherein the existence of the nucleotide sequence indicates the presence of Francisella tularensis in the sample.
  • a nucleotide detection method such as PCR and flurogenic 5' nuclease PCR assay
  • nucleotide sequences located on different loci of the Francisella tularensis bacterium genome and primers and the hybridization probes used in detecting the specific nucleotide sequences. Also disclosed is a method for identifying Francisella tularensis by analyzing samples taken from monitoring devices, such as air monitors, for the nucleotide sequences that are specific to Francisella tularensis.
  • nucleotide sequences that have been identified are unique to the Francisella tularensis bacterium, using the primers and hybridization probes to detect the presence of the Francisella tularensis bacterium is far more reliable than existing methods and partly reduces the occurrence of false positive and false negative results.
  • Francisella tularensis is the bacterium that causes Tularemia, a disease that can be fatal if not detected and treated with appropriate antibiotics.
  • the symptoms of Tularemia “could include sudden fever, chills, headaches, muscle aches, joint pain, dry cough, progressive weakness, and pneumonia", this information can be found at the internet address www.bt.cdc.gov/documentsapp/FactSheet/Tularemia/about.asp. It is on the Center for Disease Control and Prevention (CDC) list of possible bacteria that has potential as a biological warfare weapon.
  • the CDC has developed a list of possible pathogens that may be used as weapons of mass destruction.
  • Francisella tularensis has been listed in Category A of possible diseases and agents.
  • a key element in developing defenses against the use of Francisella tularensis is the ability to quickly and accurately detect the presence of the bacterium. Early detection will allow for the implementation of effective counter measures. Additionally, early detection will allow for the identification and treatment of those that may have been exposed to the bacterium. Early detection and treatment is essential for the treatment of Tularemia because although the disease may be fatal, it is usually treatable with antibiotics upon early detection. [00013] Existing detection methods have resulted in a higher than acceptable rate of false positive and false negative results. Such results are inadequate and can create confusion regarding the appropriate countermeasures, if any, that should be undertaken because it is unclear whether the bacterium is present or not. If the bacterium is not present, undertaking counter measures may cause undue expense and create unwarranted concern among those that may incorrectly believe they have been exposed.
  • a typical assay can determine the presence of SEQ ID Nos 4 and 8 using the sequence specific primers and the hybridization probes. If there is a positive result for the presence of Francisella tularensis then an assay is run to determine the presence of additional amplicon sequences, as a means to double check for the presence of Francisella tularensis.
  • PCR is a technique utilized to amplify genomic DNA. Typical PCR reactions include appropriate PCR buffers, nuclease polymerase and one or more oligonucleotide primers and hybridization probes. Various modifications of PCR techniques are possible as detailed in Current Protocols in Molecular Biology ed. F.M. Ausubel, R. Brent, D.D. Moore, K. Struhle, Massachusetts General Hospital and Harvard Medical School (1987) which is hereby incorporated by reference. The following US patents describe PCR and are incorporated herein by reference: US 4,683,195; US 4,683,202; US 4,800,159.
  • TaqMan® One method that may be used for real-time PCR amplification and detection is TaqMan®.
  • the principles involved in the conventional Taqman® 5' exonuclease assay are described in detail by Holland et al in, Detection of specific polymerase chain reaction product by utilizing the 5' — 3' exonuclease activity of Thermus aquaticus polynucleotide polymerase, Proc Natl Acad Sci U S A 88 (16):7276-80, 1991, which is herein incorporated by reference.
  • TaqMan® real time detection can also be used to simultaneously detect a plurality of nucleic acid targets when it is used with multiplex PCR, which enables simultaneous detection of more than one target sequence, thus enhancing detection accuracy.
  • a few examples of typical PCR instruments include the ABI prism 7700, the Cepheid Smart Cycler, and the Bio-Rad iCycler.
  • the sample In order to use a PCR assay method for detection of the Francisella tularensis bacterium, the sample must be prepared to extract all DNA that may be present. The following is a protocol for the preparation of samples taken from ambient air monitoring devices for nucleotide detection using fluorogenic 5' nuclease PCR assay.
  • This part of the assay protocol is performed in segregated work areas and in a biosafety cabinet using BSL 2 practices.
  • NTC no template control
  • Taq. 5 Spin Cepheid tubes in Cepheid microfuge for about 4 seconds. This mixes the PCR reaction mix and DNA into the optic diamond area. Check to see that the optic area is filled.
  • CT values from 34 to 35 indicate negative readings - no Francisella DNA detected.
  • CT values below 34 indicate positive readings - Francisella DNA detected.
  • Table 1 shows the results of assay runs that were performed using the above described protocol.
  • An assay set containing the primers and probe for each amplicon sequence was added to a sample containing either the Francisella tularensis bacteria or Francisella philomiragia.
  • the Francisella philomiragia bacteria is similar in genetic composition to its cousin Francisella tularensis.
  • Francisella philomiragia was, therefore, used as a control to demonstrate that the primers and probe derived from the Francisella tularensis specific amplicon could distinguish between genetic near neighbors, thereby demonstrating the specificity of the amplicon sequence.
  • Three different strains of the philomiragia bacteria were used, 25016, 25017 and 25018.
  • the Francisella tularensis and the Francisella philomiragia DNA was obtained form American Type Culture Collection.
  • the assay sets containing the probes and primers were obtained from various vendors such as ABI and Biosearch.
  • nucleotide sequences disclosed herein are specific to Francisella tularensis.
  • air monitors are an effective method of obtaining samples for analyses
  • a wide variety of other media and methods may be used to provide the samples for analysis for the Francisella tularensis bacterium.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne l'identification de séquences nucléotidiques spécifiques à Francisella tularensis, servant de marqueur ou de signature pour l'identification de cette bactérie. L'invention concerne, de plus, des amorces avant et inverses et des sondes d'hybridation dérivées de ces séquences nucléotidiques, utilisées dans des méthodes de détection de nucléotides pour détecter la présence de la bactérie.
PCT/US2003/024218 2002-08-01 2003-07-31 Sequences nucleotidiques specifiques a francisella tularensis et methodes de detection de francisella tularensis WO2004013357A2 (fr)

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US40089202P 2002-08-01 2002-08-01
US60/400,892 2002-08-01
US10/630,154 US7172868B2 (en) 2002-08-01 2003-07-29 Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis
US10/630,154 2003-07-29

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WO2009151982A1 (fr) * 2008-05-30 2009-12-17 Ibis Biosciences, Inc. Compositions pour utilisation dans l’identification de francisella
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8017358B2 (en) 2001-03-02 2011-09-13 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US8026084B2 (en) 2005-07-21 2011-09-27 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
US8088582B2 (en) 2006-04-06 2012-01-03 Ibis Biosciences, Inc. Compositions for the use in identification of fungi
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8158936B2 (en) 2009-02-12 2012-04-17 Ibis Biosciences, Inc. Ionization probe assemblies
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US8173957B2 (en) 2004-05-24 2012-05-08 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US8182992B2 (en) 2005-03-03 2012-05-22 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US8187814B2 (en) 2004-02-18 2012-05-29 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US8214154B2 (en) 2001-03-02 2012-07-03 Ibis Biosciences, Inc. Systems for rapid identification of pathogens in humans and animals
US8268565B2 (en) 2001-03-02 2012-09-18 Ibis Biosciences, Inc. Methods for identifying bioagents
US8298760B2 (en) 2001-06-26 2012-10-30 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8407010B2 (en) 2004-05-25 2013-03-26 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8550694B2 (en) 2008-09-16 2013-10-08 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods
US8563250B2 (en) 2001-03-02 2013-10-22 Ibis Biosciences, Inc. Methods for identifying bioagents
US8822156B2 (en) 2002-12-06 2014-09-02 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8871471B2 (en) 2007-02-23 2014-10-28 Ibis Biosciences, Inc. Methods for rapid forensic DNA analysis
US8950604B2 (en) 2009-07-17 2015-02-10 Ibis Biosciences, Inc. Lift and mount apparatus
US9080209B2 (en) 2009-08-06 2015-07-14 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection
US9149473B2 (en) 2006-09-14 2015-10-06 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens
US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification
US9393564B2 (en) 2009-03-30 2016-07-19 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods
US9416409B2 (en) 2009-07-31 2016-08-16 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US9719083B2 (en) 2009-03-08 2017-08-01 Ibis Biosciences, Inc. Bioagent detection methods
US9758840B2 (en) 2010-03-14 2017-09-12 Ibis Biosciences, Inc. Parasite detection via endosymbiont detection
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TWI383664B (zh) * 2008-08-12 2013-01-21 Unihan Corp 支援同軸電纜多媒體聯盟標準的電子裝置
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AU2003269938A8 (en) 2004-02-23
AU2003269938A1 (en) 2004-02-23
US20060040268A1 (en) 2006-02-23
US7172868B2 (en) 2007-02-06
US7494778B2 (en) 2009-02-24
WO2004013357A3 (fr) 2004-05-13

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